A consensus sequence for binding of SmcR, a Vibrio vulnificus LuxR homologue, and genome-wide identification of the SmcR regulon

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. In the present study, DNA targets of SmcR, a Vibrio vulnificus LuxR homologue, were selected from a random pool of DNA fragments by using a cycle selection procedure consisting of in vitro DNA- SmcR interaction, purification of SmcR- DNA complexes, and PCR amplification of SmcR- bound DNA. The amplified DNA fragments were cloned and analyzed separately by electrophoretic mobility shift assay to verify the specific binding of SmcR to the DNA. The DNA sequences bound by SmcR were determined by DNase I footprinting, and alignment of the resulting 29 sequences revealed a 22- bp consensus SmcR- binding sequence, 5'- TTATTGATWWRWTWNTNAATAA-3' (where W represents A or T, R is G or A, and N is any nucleotide), with an 8- bp ( TTATTGAT) inverted repeat. The consensus sequence revealed greater efficiency for the binding of SmcR than the SmcR- binding sequence previously identified within PvvpE. Mutational analysis demonstrated that the 9th and 10th bases from the center are the most essential for SmcR binding. A genome- wide search using the consensus sequence predicted that at least 121 genes are under the control of SmcR, and 10 of these newly identified SmcR regulon members were verified as being regulated by SmcR in V. vulnificus as well as in vitro. The consensus sequence and newly identified genes should be of use for elucidating the regulatory mechanism of SmcR and provide further insight into the role of the quorum sensing in V. vulnificus pathogenesis.